T lymphocytes have long been known to play a principal role in the regulation of the immune response. The T lymphocytes can be roughly divided into three compartments; the T inducer (helper) cells, cytotoxic T lymphocytes (CTL) and suppressor T cells. The activation of T lymphocytes occurs upon interaction with cells bearing appropriate antigen in the context of major histocompatibility complex (MHC) proteins. Specificity of the T cell response is conferred by the polymorphic, antigen-specific T cell receptor (TCR). In addition to the antigen specific TCR, a number of other cell surface proteins regulate T cell activation and impart sensitivity and plasticity to the immune response. These include the surface antigens CD2, CD4 CD8 and lymphocyte function associated antigen. Such surface antigens are non-polymorphic molecules that not only increase the avidity with which a T cell interacts with its antigen presenting cell (APC) or target cell but also may play a role in signal transduction. The CD4 and CD8 molecules are expressed on mutually exclusive populations of mature T cells that bear TCRs specific for antigen in association with MHC class II and MHC class I proteins, respectively. These molecules appear to enhance the avidity with which a T cell binds with its antigen bearing or target cell and may also promote the interaction of the TCR with its appropriate antigen. Bierer et al, "The Biologic Roles of CD2, CD4 and CD8 in T-cell Activation," Ann. Rev. Immunol., 7:579-99 (1989).
The phenomenology of MHC-restricted T lymphocyte interactions has previously been well documented. Cells of the T helper/inducer subset will generally recognize antigen only in association with class II major histocompatibility (MHC) gene products. Recognition occurs on the surface of antigen presenting cells and accounts for the MHC genetic restriction of antigen recognition. Cytotoxic T lymphocytes (CTL), which are well known effector cells in transplantation immunity, viral infection and certain anti-tumor responses, generally recognize antigen in association with class I MHC gene products. While the rules governing the activation of MHC-restricted T cells, and particularly of class II MHC-restricted T cells have been well described, the underlying mechanisms are still being defined.
Most of the understanding in the art of T lymphocyte biology has come from in vivo studies and in vitro experiments using heterogeneous T cell populations. Such methodologies have certain inherent limitations, including low frequency of specific T lymphocytes and the potential for multicellular complex interactions. More recently, a better understanding of T cell activation and of the factors involved has allowed for the isolation and propagation of T cell lines and clones of defined specificity. Two basic strategies have been devised. One approach is to clonally expand and propagate normal immune T lymphocytes through the repetitive stimulation with the appropriate antigen, with or without the presence of growth factors. The second approach parallels the approach that has been utilized to produce monoclonal antibodies from B cell hybridomas prepared by cell hybridization of antibody-producing B cells and myeloma cells, and involves the immortalization of T lymphocytes through somatic cell hybridization with T cell lymphomas.
The latter methodology has been used extensively for the production of antigen-specific, class II MHC-restricted, T-T hybridomas, which have several advantageous properties. See, e.g. Kappler, et al, "Antigen inducible, H-2 restricted, interleukin-2 producing T cell hybridomas. Lack of independent antigen and H-2 recognition", J. Exp. Mede, 153:1198; (1981), Rock, K. L., "Functional T-cell Hybridomas, in Hybridoma Technology in the Biosciences and Medicine.," Ed. T. A. Springer, Plenum Press, N. Y. (1985). These hybridomas have essentially limitless growth potential and grow readily under standard culture conditions. Furthermore, the immunological activity of these hybridomas is not subject to cyclic fluctuations, as is seen with restimulated normal clones. The T-inducer hybridomas produce lymphokines in response to T cell receptor (TCR) stimulation, which provides a sensitive and quantitative assay to measure cellular activation. Consequently, the class II MHC-restricted T-T hybridomas have proven to be particularly useful in the analysis of the structure and function of surface receptors, cell-cell interactions and T cell activation involving T helper cells. See, for example, Rock, K. L., "Functional T-cell Hybridoma", in Hybridoma Technology in the Biosciences and Medicine, Ed. T. A. Springer, Plenum Press, N. Y. (1985); Marrack, P. and J. Kappler. "The antigen-specific, major histocompatibility complex-restricted receptor on T cells." Adv. Immunol. 38:1 (1986), Bierer, B. E., et al, "The biologic roles of CD2, CD4, and CD8 in T cell activation." Ann. Rev. Immunol. 7: 579 (1989). Moreover, since the T-inducer hybrids do not generally require costimulatory signals, it has been possible to stimulate them with fixed or disrupted antigen presenting cells as well as model membrane systems. Shimonkevitz, R. J. et al, "Antigert recognition by H-2 restricted T cells. Cell-free antigen processing," J. Exp. Med. 158:303, (1983), Watts, T. H. et al, "Antigen presentation by supported planar membranes containing affinity purified IA.sup.d.) Proc. Natl. Acad. Sci. USA 81:1883 (1984). Therefore, antigen-specific class II MHC restricted T-T hybridomas have provided a powerful and useful tool in analyzing the events in antigen presentation by providing an assay to follow the appearance of antigen-MHC class II molecule complexes.
In contrast to the above results, there has been only limited success in generating antigen-specific class I MHC restricted T cell hybridomas, particularly with the most commonly used murine fusion partner, the BW5147 thymic lymphoma. Heretofore, such fusions with BW5147 have generally been unproductive. The reason why such hybrids have not been easily obtained from fusions with BW5147 is not known.
Despite the foregoing, there have been isolated reports of successful generation of class I MHC-restricted hybridomas with BW5147. Whitaker et al, for example, have described the preparation of H-2 restricted, reovirus-specific cytotoxic T cell hybrids using BW5147 as a fusion partner. "CT hybridomas: Tumor Cells Capable of Lysing Virally Infected Target Cells," Journal of Immunology, vol. 129, no. 2 pp. 900-903. These hybrids, however, require a mitogenic lectin for stimulation and do not respond well to antigen and appropriate antigen presenting cells alone. Other examples of class I MHC-restricted hybridomas generated with BW5147 cells have previously been reported by Kaufmann, Y. G. et al, "Cytotoxic T. Lymphocyte T cell hybridomas that mediate specific tumor-cell lysis in vitro.", Proc. Natl. Acad. Sci. USA, 78:2502, (1981), and by Endres, R. P. et al, "An IL-2 secreting T cell hybridoma that responds to a self class I histocompatibility antigen in the H-2D region.", J. Immunol. 131:1656 (1983). However, such hybrids are identified at low frequency and may represent "high affinity" T cells that are not CD8 dependent. See, MacDonald et al, "Clonal Heterogeneity in the functional requirement for Lyt-2/3 molecules on cytolytic T Lymphocytes (CTL): Possible implications for the affinity of CTL antigen receptors." , Immunol. Rev. 68:89 (1982). Furthermore, the successful isolation of such hybridomas has been limited to those that are MHC reactive (e.g. allo- or auto-reactive) and has not been described for antigen-specific, MHC-restricted cells.
Other fusion partners have been employed to generate class I MHC-restricted T-T hybrids, but such cells have not achieved widespread usage. Accordingly, there exists a need for an immortalized fusion partner which will allow for the successful generation and detection of class I, MHC-restricted T-T hybridomas, including those that are CD8 dependent.
In those instances where somatic hybridization technology has been used to establish functional class I MHC-restricted T cell hybrids, screening of the clones for responsiveness to class I MHC alloantigens or to antigen plus class I MHC proteins has typically been conducted using a conventional chromium release assay. This assay is routinely employed in the study of cytotoxic T cells and involves incubation of serial dilutions of cytolytic effector cell populations in the presence of a constant number of .sup.51 Cr labeled target cells at low effector to target ratios. At these low ratios, each effector cell is saturated with targets and kills at maximum efficiency. After a prescribed period of time, the radioactivity released into the supernatants is measured and the percent specific cytotoxicity can be calculated.
Since what is being measured in the chromium release assay is the cytolytic capability of the effector cells, this assay necessarily requires the use of intact target cells. This requirement constrains the experimental manipulation of target cells, for example with drugs or chemical reagents. Moreover, intact cells have a tendency to retain the isotope and hence, measurement of the isotope release does not completely reflect the strength of the CTL response. Another disadvantage of the chromium release assay is that it is labor intensive and presents the potential of health and safety risks. In view of all of the foregoing, an alternative assay for screening for the cytotoxic fusion product and for studying antigen presentation in association with class I MHC-restricted molecules is indicated.
Although it has been observed that many cytotoxic T lymphocytes have the capacity to produce lymphokines in response to antigen, they generally produce lower levels of lymphokines such as IL-2. Accordingly, this capability has not been employed in the study of the events of class I MHC-restricted antigen presentation and cell-cell interaction.
Accordingly, it is an object of the present invention to provide a novel BW5147 fusion partner for the generation and detection of stable, functional, class I MHC-restricted T-T hybridomas.
Another object of the present invention is to provide a BW5147 fusion partner which is permissive for lymphokine production in class I MHC-restricted T-T hybridomas.
A still further object of the present invention is to generate lymphokine producing, class I MHC-restricted T-T hybridomas using BW5147 as a fusion partner.
Yet another object of the present invention is to provide a quantitative and sensitive assay for the generation and detection of lymphokine-producing, class I MHC-restricted hybrids.
These as well as other objects and advantages are accomplished by the present invention, the nature and scope of which will become apparent from the following specification.